Two major developments in the practice of chromatographic separations have been of dramatic importance over the last decade or so in facilitating the isolation of natural products, separation of components of mixtures, and analysis of complex compositions. These are the proliferation of the variety of available ligands for affinity chromatography, wherein the separation or analysis depends on the specific interaction between a supported ligand and a desired analyte, and the advent of high performance liquid chromatography (HPLC) which permits rapid and efficient separation of multiple components. These developments have overlapped only to a limited extent, as HPLC generally utilizes conditions which are inimical to many of the legends used as specific affinity partners. The most common affinity partner for use in these techniques with respect to a spectrum of possible analytes has been specific immunoglobulins or immunoreactive fragments thereof. In general, this type of ligand is unstable with respect to the conditions employed in HPLC. HPLC often employs nonaqueous solvents, which are denaturing to many affinity ligands and the high pressures employed are also destructive to many of these substances.
In affinity based chromatography, a variety of solid supports and of affinity ligands can be used, as summarized in an early review article by May, S. W. in Separation and Purification 3rd Ed. (1978) Edmond S. Perry, et al, ed., vol. 12 in Techniques of Chemistry (J. Wiley). This review describes suitable supports for affinity chromatography emphasizing polysaccharide supports in addition to polyacrylamide gels, mixed gels, and various glasses and silica derivatives. Of these, only silica derivatives have gained wide acceptance for use in HPLC. However, the extent of derivatization of the support to modify its binding characteristics has been limited to altering hydrophobicity by conjugation of various hydrocarbon ligands or other simple molecules.
The present invention enables a convenient crossover between the HPLC and affinity approaches by providing ligands which have the required affinity specific for a selected member of an array of possible analytes with capability to withstand the conditions of HPLC.
Others have attempted this crossover in various ways. Peterson, E. A. et al Meth Enz (1984) 104:113-133 describe "displacement" chromatography wherein competition for the adsorption sites between adsorbed components is substituted for competition with eluant. Chromatographic supports which employ carbohydrates, such as cyclodextrins, with differential specific affinities for the substances to be separated have also been reported (Armstrong, D. W. et al J Chrom Sci (1984) 22:411-415.
The ligands employed in the invention method are peptides of 4-20 amino acids which are designated "paralogs" herein. A paralog mimics the portion of an immunoglobulin which specifically binds to the antigenic determinant or epitope of the antigen to which the antibody is raised. The segment complementary to this epitope is commonly designated a paratope, and since the peptide sequence in the paralog need not be the same as that occurring in the raised antibodies, the term paralog (or paratope analog) is used.
Synthesis of, and identification of, paralogs has been done previously to a very limited extent. Atassi, M. Z., et al J Biol Chem (1977) 252:8784-8787 described the specific design of a peptide complementary to the antigenic sites of lysozyme. Knowledge of the three-dimensional contours of lysozyme permitted the synthesis of a peptide of dimensions and electron density patterns analogous to the deduced determinant. The paralog was obtained by preparing a peptide sequence deliberately complementary in dimension and electron distribution to the determinant-mimicking peptide. The pseudo "paratope" peptides inhibited the reaction of lysozyme with antisera and specifically bound lysozyme to the exclusion of myoglobin or antibody. Later work from the same group resulted in the synthesis of a peptide representing the acetyl choline binding site of a specific receptor and of a binding site in trypsin (McCormick, D. J., et al Biochem J (1984) 224:995-1000; Atassi, M. Z. Biochem J (1985) 226:477-485). The paralog (or analogous receptor- or enzyme binding site-mimicking) peptides were based on known parameters associated either with the antigenic determinant or with the determinant binding moiety.
Recent work has shown that the idiotypic surface of antibodies can be mapped and peptides mimicking portions of this surface can be prepared. As expected, the idiotopes and paratopes do not precisely coincide. Seiden, M. V. Am Assoc Immunol (1986) 136:582-587; Roux, K. H. et al Proc Natl Acad Sci USA (1987) 84:4984-4988.
Recently, methods to mimic epitopes as specifically binding complementary components without knowledge of the characteristics of the specific interaction have been disclosed. The most relevant work is that of Geysen, H. M. at the Commonwealth Serum Laboratories in Australia. Geysen has devised an empirical method for preparing a panel of multiple candidate sequences whose ability to bind specifically to antibody can be empirically tested. In the Geysen approach, each of the candidate peptides is separately synthesized on an individual polyethylene support rod in relatively small amount. The support rods are arranged conveniently so as to dip individually into the wells of a microtitre tray. Typically 96 separate peptides can be simultaneously synthesized (the number corresponding to the arrangement of commercially available trays). The 96 peptides can also be simultaneously assayed for binding to antibodies or receptors using standard radioimmunoassay or ELISA techniques. (See, for example, Proc Natl Acad Sci (USA) (1984) 81 3998-4002, PCT applications WO86/00991 and WO86/06487.)
A variety of candidate peptides can also be simultaneously synthesized in separate containers using the T-bag method of Houghten, R., Proc Natl Acad Sci (USA) (1985) 82:5131-5135.
The performance of the paralogs may be improved in some instances by controlling their 3-dimensional conformation through the use of "molecular sticks" as described in U.S. patent application Ser. No. 172,623, filed 24 March 1988, assigned to the same assignee and incorporated herein by reference.
The foregoing elements of the art can be productively used as a resource to construct the ligands needed for the chromatographic substrates and for the conduct of the methods of the herein invention.